ADA Knouckout HCT 116 Polyclonal Cells are a CRISPR/Cas9-mediated gene disruption model in the HCT 116 human colorectal carcinoma cell line. This polyclonal knockout cell population harbors targeted loss-of-function alleles in the ADA gene, which encodes adenosine deaminase. The heterogeneous pool is generated without clonal isolation, preserving the genetic diversity of the parental line while introducing ADA gene disruption. As a polyclonal product, it provides a practical and robust tool for studying ADA function in cancer, facilitating experiments that do not depend on monoclonality.
The HCT 116 host cell line is a well-established human colorectal carcinoma model featuring a mutation in codon 13 of the KRAS proto-oncogene, leading to constitutive KRAS activation. This line is extensively used in cancer research, drug discovery, and signaling studies due to its well-characterized epithelial morphology and defined genetic background. It is particularly suited for investigating oncogenic signaling and metabolic adaptations in colorectal cancer.
ADA catalyzes the irreversible deamination of adenosine and deoxyadenosine to inosine and deoxyinosine, playing a central role in purine metabolism and nucleotide homeostasis. ADA expression is regulated by upstream factors such as NF-??B and Notch1 signaling, and in immune cells by TCR engagement. The enzyme directly interacts with CD26/DPP4 on the cell surface and indirectly modulates adenosine receptor signaling. Loss of ADA function leads to accumulation of its substrates, resulting in elevated dATP levels and altered cAMP signaling via adenosine receptors. These changes disrupt intracellular purine pools and can trigger apoptosis in lymphocytes, underscoring ADA’s importance in both metabolism and immune regulation.
In HCT 116 cells, ADA knockout enables detailed studies of purine metabolism rewiring in colorectal cancer. The buildup of adenosine and deoxyadenosine affects nucleotide pools and energy metabolism, potentially impacting KRAS-driven proliferation and survival. This model is highly relevant for investigating adenosine-mediated immunosuppression in the tumor microenvironment and for evaluating drug sensitivity to ADA inhibitors, offering insights into metabolic vulnerabilities in cancer.
Researchers can use this knockout model in diverse functional assays: Western blotting or RT-qPCR for ADA expression, LC-MS for adenosine/inosine quantification, and flow cytometry to examine ADA-CD26 interaction. Proliferation and apoptosis can be assessed by MTT/XTT or Annexin V assays, while cAMP assays probe adenosine receptor signaling. Migration and invasion assays further elucidate the role of ADA in cancer cell behavior. For additional details or to request a quote, please contact Ascent Research.
